The cultivation of agricultural crop plants serves mainly for the production of foodstuffs for humans and animals. Monocultures in particular, which are the rule nowadays, are highly susceptible to an epidemic-like spreading of diseases. The result is markedly reduced yields. To date, the pathogenic organisms have been controlled mainly by using pesticides. Nowadays, the possibility of directly modifying the genetic disposition of a plant or pathogen is also open to man.
Resistance generally describes the ability of a plant to prevent, or at least curtail the infestation and colonization by a harmful pathogen. Different mechanisms can be discerned in the naturally occurring resistance, with which the plants fend off colonization by phytopathogenic organisms. These specific interactions between the pathogen and the host determine the course of infection (Schopfer and Brennicke (1999) Pflanzenphysiologie, Springer Verlag, Berlin-Heidelberg, Germany).
With regard to the race specific resistance, also called host resistance, a differentiation is made between compatible and incompatible interactions. In the compatible interaction, an interaction occurs between a virulent pathogen and a susceptible plant. The pathogen survives, and may build up reproduction structures, while the host mostly dies off. An incompatible interaction occurs on the other hand when the pathogen infects the plant but is inhibited in its growth before or after weak development of symptoms. In the latter case, the plant is resistant to the respective pathogen (Schopfer and Brennicke, vide supra). However, this type of resistance is specific for a certain strain or pathogen.
In both compatible and incompatible interactions a defensive and specific reaction of the host to the pathogen occurs. In nature, however, this resistance is often overcome because of the rapid evolutionary development of new virulent races of the pathogens (Neu et al. (2003) American Cytopathol. Society, MPMI 16 No. 7: 626-633).
Most pathogens are plant-species specific. This means that a pathogen can induce a disease in a certain plant species, but not in other plant species (Heath (2002) Can. J. Plant Pathol. 24: 259-264). The resistance against a pathogen in certain plant species is called non-host resistance. The non-host resistance offers strong, broad, and permanent protection from phytopathogens. Genes providing non-host resistance provide the opportunity of a strong, broad and permanent protection against certain diseases in non-host plants. In particular, such a resistance works for different strains of the pathogen.
Fungi are distributed worldwide. Approximately 100 000 different fungal species are known to date. Thereof rusts are of great importance. They can have a complicated development cycle with up to five different spore stages (spermatium, aecidiospore, uredospore, teleutospore and basidiospore).
During the infection of plants by pathogenic fungi, different phases are usually observed. The first phases of the interaction between phytopathogenic fungi and their potential host plants are decisive for the colonization of the plant by the fungus. During the first stage of the infection, the spores become attached to the surface of the plants, germinate, and the fungus penetrates the plant. The chemical properties of the surface (e.g., cuticle) are an important determinant for the recognition of the plant as a potential host (for review see Tucker and Talbot (2001) Surface attachment and pre-penetration stage development by plant pathogenic fungi, Annual Review of Phytopathology Vol. 39: 385-417). Fungi may penetrate the plant via existing ports such as stomata, lenticels, hydatodes and wounds, or else they penetrate the plant epidermis directly as the result of the mechanical force and with the aid of cell-wall-digesting enzymes. Specific infection structures are developed for penetration of the plant. The soybean rust Phakopsora pachyrhizi directly penetrates the plant epidermis. After crossing the epidermal cell, the fungus reaches the intercellular space of the mesophyll, where the fungus starts to spread through the leaves. To acquire nutrients the fungus penetrates mesophyll cells and develops haustoria inside the mesophyll cell. During the penetration process the plasmamembrane of the penetrated mesophyll cell stays intact. Therefore the soybean rust fungus establishes a biotrophic interaction with soybean.
The biotrophic phytopathogenic fungi, such as many rusts, depend for their nutrition on the metabolism of living cells of the plants. This type of fungi belong to the group of biotrophic fungi, like other rust fungi, powdery mildew fungi or oomycete pathogens like the genus Phytophthora or Peronospora. The necrotrophic phytopathogenic fungi depend for their nutrition on dead cells of the plants, e.g. species from the genus Fusarium, Rhizoctonia or Mycosphaerella. Soybean rust has occupied an intermediate position, since it penetrates the epidermis directly, whereupon the penetrated cell becomes necrotic. After the penetration, the fungus changes over to an obligatory-biotrophic lifestyle. The subgroup of the biotrophic fungal pathogens which follows essentially such an infection strategy is heminecrotrophic. In contrast to a heminecrotrophic pathogen, a hemibiotrophic pathogen lives for a short period of time in a biotrophic manner and subsequently starts killing the host cell and/or host organism, i.e., changes for the rest of its life-cycle to a necrotrophic life-style.
The early, pre-penetration stages, i.e., attachment of the spore, germination of the spore, hyphal growth and the development of the infection structure (appressorium) are crucial for the success of the infection. Unfortunately the underlying molecular mechanisms are still unknown. Nevertheless there are a few indications that the expression of hydrophobins by the fungus are required for the development of hyphae and the appressorium. For a comprehensive review about the pre-penetration stage in plant-pathogen interactions and the involvement of hydrophobins see Tucker and Talbot, “Surface attachment and pre-penetration stage development by plant pathogenic fungi (Annu. Rev. Phytopathol 2001, 39:385ff).
Hydrophobins are a class of small, cysteine-rich proteins with a length of about 100-150 amino acids which occur in nature only in filamentous fungi. They are amphiphilic and can form a layer on the surface of an object. Their natural functions include inter alia the coating of fungal spores, so that these do not stick together, the coating of aerial hyphae for reducing the surface tension of water and thus for facilitating the absorption of water, and possibly the signal transmittance between a fungus and its environment (Whiteford. J. F. Spanu, P. D. (2001), Fungal Genet. Biol. 32 (3): 159-168; Wösten et al. (1999) Current Biol. 19: 1985-88; Bell et al. (1992), Genes Dev. 6: 2382-2394).
Hydrophobins generally have eight cysteine units. They can be isolated from natural sources, but can also be obtained by means of genetic engineering methods, as described for example in WO 2006/082251 and WO 2006/131564.
The first isolation and purification of hydrophobin was carried out from Schizophyllum commune in 1999. In the meantime, hydrophobin genes have been identified in Ascomycetes, Deuteromycetes and Basiodiomycetes. Some fungi comprise more than one hydrophobin gene, e.g. Schizophyllum commune, Coprinus cinereus and Aspergillus nidulans. On the basis of differences with regard to the hydropathy and the biophysical properties of the hydrophobins, these have been divided into two categories: class I and class II. Complementation experiments have shown that hydrophobins of the one class are able to replace hydrophobins of the other class to a certain degree as far as function is concerned. The different hydrophobins appear to be involved in different fungal development stages and to perform different functions therein (van Wetter et al. (2000) Mol. Microbiol. 36:201-210; Kershaw et al. (1998) Fungal Genet. Biol. 23:18-33).
Soybean rust has become increasingly important in recent times. The disease may be caused by the biotrophic rusts Phakopsora pachyrhizi (Sydow) and Phakopsora meibomiae (Arthur). They belong to the class Basidiomycota, order Uredinales, family Phakopsoraceae. Both rusts infect a wide spectrum of leguminosic host plants. P. pachyrhizi, also referred to as Asian rust, is the more aggressive pathogen on soy (Glycine max), and is therefore, at least currently, of great importance for agriculture. P. pachyrhizi can be found in nearly all tropical and subtropical soy growing regions of the world. P. pachyrhizi is capable of infecting 31 species from 17 families of the Leguminosae under natural conditions and is capable of growing on further 60 species under controlled conditions (Sinclair et al. (eds.), Proceedings of the rust workshop (1995), National SoyaResearch Laboratory, Publication No. 1 (1996); Rytter J. L. et al., Plant Dis. 87, 818 (1984)). P. meibomiae has been found in the Caribbean Basin and in Puerto Rico, and has not caused substantial damage as yet.
P. pachyrhizi can currently be controlled in the field only by means of fungicides. Soy plants with resistance to the entire spectrum of the isolates are not available. When searching for resistant plants, six dominant genes Rpp1-5 and Rpp?(Hyuuga), which mediate resistance of soy to P. pachyrhizi, were discovered. The resistance was lost rapidly, as P. pachyrhizi develops new virulent races.
In recent years, fungal diseases, e.g. soybean rust, has gained in importance as pest in agricultural production. There was therefore a demand in the prior art for developing methods to control fungi and to provide fungal resistant plants.
Much research has been performed on the field of powdery and downy mildew infecting the epidermal layer of plants. However, the problem to cope with soybean rust which infects the mesophyll remains unsolved.